Sphingolipids are a conserved family of lipids built upon a sphingoid base backbone. Sphingolipids serve structural membrane functions, whereas their metabolism produces signaling molecules involved in regulating mammalian development, immune function, inflammation and cellular stress responses. Studies supported by this grant have explored the role of sphingolipids in the model organism Drosophila melanogaster. These studies have resulted in the chemical characterization of Drosophila sphingoid bases and the identification of novel endogenous Drosophila sphingolipids with potent growth-inhibitory activity. Further, we have shown that Sply mutants lacking expression of Drosophila sphingosine-1-phosphate (S1P) lyase (SPL), which is responsible for the final step of sphingolipid degradation, accumulate sphingolipid intermediates and develop a progressive myopathy in the thoracic muscles needed to power flight. The Sply myopathy is corrected by reducing sphingolipid production, indicating sphingolipid intermediates play a causative role. We have observed that mutants in key membrane proteins such as dynamin and dystroglycan phenocopy the Sply myopathy. We have also conducted cell-based investigations that suggest that Sply/SPL is required for normal AKT signaling, protein translation, myoblast fusion, myoblast gene expression and control of autophagy. Many of these interactions have been corroborated in murine C2C12 cells, indicating that SPL plays a conserved role in myoblast survival, fusion and differentiation. We have also found that sphingosine kinase and S1P lyase are dynamically upregulated during murine muscle regeneration, leading to a transient peak in S1P levels in regenerating muscle. These collective observations have led us to propose our central hypothesis, which states that SPL plays a critical role in muscle biology, development and homeostasis. The specific aims of our proposal are, thus: 1) To define the role of SPL in muscle cell biology; 2) To dissect the role of SPL in muscle development; and 3) To establish the role of SPL in muscle atrophy and regeneration. Our long-term goals are to exploit Drosophila to elucidate the role of sphingolipids in biology, membrane function, and tissue homeostasis and to define the relevance of these findings to human disease. Due to species-specific structural differences in sphingolipids and the lack of S1P receptors in lower eukaryotes, we do not expect the two systems to be equivalent. Thus, our goals are to compare the biochemical and molecular events associated with SPL loss in Drosophila and murine cell and animal models, with the intent of developing a comprehensive understanding of how SPL functions in the context of muscle tissue and how its function may have been modified throughout evolution. These studies should be readily achieved by our team, which has extensive experience in sphingolipid biochemistry, Drosophila genetics, and the genetics and pathology of MD.